Synthetic RNAs are a promising new class of therapeutics for non-virus-mediated gene therapy, vaccines and protein replacement therapeutics, as well as in immuno-oncology and personalized cancer vaccines (Sahin et al., (2014): Nature Reviews Drug Discovery 13, 759-80; Weissman, (2015), Expert Review of Vaccines, 14(2):265-81). Synthetic RNAs are commonly manufactured by in vitro transcription (IVT) of a DNA template that encodes the antigen or protein of interest (Sahin et al., (2014); Steinle et al., (2017), Stem Cells, 35(1):68-79).
One limitation associated with the therapeutic use of synthetic RNA is an immunostimulatory response induced by double-stranded RNA (dsRNA) contaminants created during IVT (Devoldere et al., (2016), Drug Discover Today, 21(1), 11-25; Loomis et al., (2016), Journal of Materials Chemistry 4:1619-32; Triana-Alonso et al., (1995), The Journal of Biological Chemistry, 270 (11): 6298-6307). The immunostimulatory response of cells results from activation of receptors that trigger secretion of interferons and inflammatory cytokines (Devoldere et al., (2016); Loomis et al., (2016); Kariko et al., (2005) Immunity, 23:165-175).
Several methods have been developed to separate desired IVT products from contaminating dsRNA. These include various chromatography techniques such as: ion exchange high performance liquid chromatography (HPLC), reverse phase HPLC, hydrophobic interaction HPLC, low or normal pressure liquid chromatography, size exclusion chromatography, oligo dT affinity chromatography, and core bead chromatography (Kariko et al., (2011) Nucleic Acids Research, 39 (21), e142; Weissman, et al., (2012) Methods in Molecular Biology, 969:43-54; Koubek et al., (2013) RNA, 10:1449-59; US 2016/0024141; US 2016/0024140 A1; U.S. Pat. No. 8,383,340; WO 2014/144711; US 2016/0032316; WO 2014/144767; US 2016/0326575). Enzymatic digestion of dsRNA with RNase III, RNase V1, Dicer, and Chipper is also implemented to reduce dsRNA (US 2016/0032316).
The use of a physical separation method to remove the dsRNA from IVT reactions increases the cost and labor involved in the production of IVT RNAs that minimally activate innate immune responses.